DA rats from two colonies differ genetically and in their arthritis susceptibility
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- Rintisch, C. & Holmdahl, R. Mamm Genome (2008) 19: 420. doi:10.1007/s00335-008-9125-x
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The arthritis-susceptible DA rat is one of the most commonly used rat strains for genetic linkage analysis and is instrumental for the identification of many genetic loci. Even though DA rats were kept as inbred lines at different institutes and suppliers, it became obvious that the various breeding stocks differed genetically. To be able to compare the results from different linkage studies it is very import to verify the genetic background of the substrains used in those studies. We performed a genetic and phenotypic analysis of two DA substrains, DA/ZtmRhd and DA/OlaHsd, and found several genetic differences. One of the allelic differences between the DA/ZtmRhd and the DA/OlaHsd strain was located at rat chromosome 3, a 17-Mb large fragment, including the peak marker of a previously identified quantitative trait locus (QTL) for collagen-induced arthritis, Cia11. In addition, the substrains exhibited a significant difference in the susceptibility to pristane-induced arthritis (PIA) and disease severity of collagen-induced arthritis and PIA. However, by generating and testing a congenic line, we could demonstrate that phenotypic differences were not due to the contaminating fragment on chromosome 3. Nevertheless, we conclude that DA substrains show distinct genetic differences and caution should be taken when comparing arthritis data from different DA substrains.
Rheumatoid arthritis (RA) is a chronic autoimmune disease that affects 0.5–1% of most populations, with a higher propensity in females (Alamanos and Drosos 2005). RA is a multifactorial disease of unknown etiology but with a strong genetic component (Dieude and Cornelis 2005), and recent studies based on various gene-finding strategies have identified the first genes implicated in the susceptibility to RA (Plenge et al. 2007; The Wellcome Trust Consortium 2007; Worthington 2005). However, many susceptibility genes still remain unknown. Among the difficulties involved in gene identification in complex traits are factors such as variable penetrance, variable relative risk associated to the disease allele, epistasis, genetic heterogeneity of the human population, and complex interactions between environmental and genetic factors (Lander and Schork 1994).
Despite the recent advances in human genetics, animal models of RA remain attractive tools, not only to overcome genetic complexity, but also to permit studies under stable environmental conditions (Holmdahl 2003). Experimental animal models can be used for gene identification, but more important they allow the functional characterization of arthritis-regulating loci and genes, which consequently could result in the development of novel drugs or targets for human RA. Rat models are particularly useful because large numbers of progeny can be generated from inbred strains that differ in their susceptibility to various arthritis models, including collagen-induced arthritis (CIA), adjuvant-induced arthritis (AIA), oil-induced arthritis (OIA), and pristane-induced arthritis (PIA) (Holmdahl et al. 2001). To date, linkage analysis studies in rat models of RA have identified more than 20 loci that are either specific for the given animal model or shared with other arthritis models or with RA in humans (Joe 2006).
The DA rat strain is highly susceptible to most of the experimentally induced models of arthritis and was therefore chosen as the arthritis-susceptible parental strain in a large number of conducted genetic linkage studies. Nevertheless, caution should be taken when comparing physiologic, biochemical, or genetic data among studies in which the same strains but from different breeding facilities have been used. In this study we aimed at characterizing the genetic and phenotypic differences between the two most commonly used DA substrains (DA/Ztm and DA/OlaHsd) in arthritis linkage studies. This approach provided additional information and a better understanding of previously identified quantitative trait loci (QTL) in those strains. Our original colony of inbred DA rats (referred to as DA/ZtmRhd) was established from DA/Ztm breeding pairs and we also used DA rats from Harlan Europe (referred to as DA/OlaHsd). We performed a genetic and phenotypic analysis of DA/ZtmRhd and DA/OlaHsd rats and report here several phenotypic and genetic differences between these strains.
Material and methods
DA/ZtmRhd rats were kindly provided by Prof. H.J. Hedrich in 1996 from the Zentralinstitut fuer Versuchstierkunde (Hannover, Germany) and were bred for more than 20 generations of brother-sister mating in the animal facility in Lund. DA/OlaHsd rats originating from Harlan Europe (Netherlands) were kept and bred as brother-sister matings for more than five generations in the animal facility in Lund in a climate-controlled environment with 12-h light/dark cycles. In the same facility breeding was performed to produce DA.DA/ZtmRhd-Cia11 (≥N3) congenic rats. Shortly, one DA/ZtmRhd male and two DA/OlaHsd rats were used to produce the F1 generation. Six F1 females were further backcrossed with DA/OlaHsd male rats. After three to four backcross generations, Cia11 heterozygous rats were then intercrossed and only strictly littermate-controlled experiments were performed to ensure that all phenotypic differences originated exclusively from the genetic differences in the Cia11 region. Rats were housed in polystyrene cages containing wood shavings and fed standard rodent chow and water ad libitum. The rats were free from common pathogens including Sendai virus, Hantaan virus, corona virus, reovirus, cytomegalo virus, and Mycoplasma pulmonis. All experiments had been approved by the local ethical committee (Malmö/Lund, Sweden).
Whole-genome scan of DA/ZtmRhd and DA/OlaHsd rats using SSLP markers and regions with clusters of polymorphic SNPs between DA/HanKini and DA/OlaHsd rats
Different SSLP allelesa/all SSLP tested
Additional SSLP marker
Regions with polymorphic SNPsb in Mb
Induction and evaluation of arthritis
Lathyritic collagen II (CII) was purified from Swarm rat chondrosarcoma, grown in male rats receiving β-aminopropionitrile monofumaratic salt in drinking water during the tumor-growing period as previously described (Miller and Rhodes 1982; Smith et al. 1975). Collagen-induced arthritis (CIA) was induced by a single intradermal injection of 75 μg lathyritic rat collagen II dissolved in 50 μl 0.1 M acetic acid and emulsified in 50 μl incomplete Freund’s adjuvant (IFA). Pristane-induced arthritis (PIA) was induced by a single intradermal injection of 50 μl pristane (2,6,10,14-tetramethylpentadecane, ACROS Organics, Geel, Belgium) at the base of the tail. Arthritis was induced in age- and cage-matched rats at the age of 7–9 weeks, and arthritis development was monitored in all four limbs using a macroscopic scoring system. Briefly, one point was given for each swollen and red toe, one point for each affected midfoot, digit, or knuckle, and five points for a swollen ankle (maximum score per limb was 15 and maximum score per rat was 60). The rats were examined in a blinded fashion every second to third day for one month after induction of the disease (Holmdahl 1997). All experiments in congenic rats were carried out with DA/OlaHsd littermate controls originating from F1 intercross between heterozygous congenic rats to ensure that all phenotypic differences in the congenic animals originated exclusively from the genetic differences in the chromosome 3 region.
Blood sampling and detection of antibodies
Peripheral blood was collected from rats after CIA induction at termination day by cutting the tip of the tail. The blood was left for 1 h at 4°C and centrifuged at 13,000 rpm for 10 min. Serum samples were frozen at −20°C until use. For detection of anti-collagen II antibodies (a-CII) in Eu3+LISA, plates were coated with pepsin-digested CII and incubated overnight at 4°C. After blocking with 2% BSA, diluted serum samples were added to the plates and incubated. Thereafter, biotin-labeled mouse anti-rat antibodies (anti-IgG1, anti-IgG2a, anti-IgG2b, anti-IgG2c, PharMingen, BD Bioscience, San Jose, CA, USA; polyclonal anti-rat IgG; Zymed, San Francisco, CA, USA) were added and incubated. Then Europium-labeled streptavidin (in Assaybuffer, Wallac Oy, Turku, Finland) was added. For final detection, Enhancement Solution (Wallac) was added and fluorescence emissions were read using Victor/Wallac (Wallac).
The Statview software program was used for all statistical analyses. Incidence of arthritis was analyzed by Fisher’s exact test. The nonparametric Mann-Whitney U test (comparison of two groups) or the Kruskal-Wallis test (comparison of three groups) was used in all other statistical analyses. p values less than 0.05 were considered significant.
Genetic comparison between DA/OlaHsd and DA/ZtmRhd rats
First we determined the degree of genetic heterogeneity of the two most commonly used DA substrains for linkage studies of arthritis. We therefore performed a whole-genome scan of DA/OlaHsd and DA/ZtmRhd rats using 248 well-established SSLP markers covering all 20 autosomal chromosomes and the X chromosome and compared the allele sizes in the two substrains. The marker selection was based on previous results from crosses between E3/ZtmRhd and DA/ZtmRhd rats and the E3 strain also was included in the typing as an internal control. As expected, all 248 initially typed markers were found in homozygous form in all three strains and were polymorphic between E3 and DA/ZtmRhd rats. However, we also found markers that differed in allele size between DA/ZtmRhd and DA/OlaHsd rats. As shown in Table 1, 35 of 248 markers were found to be polymorphic between the two DA substrains. This means that calculated on those markers, the two substrains differed in 14.1% of their genome. When analyzing markers from each rat chromosome (RNO) alone, the variability ranges from 0% (RNO7, 9, 15, 16) to 20% (RNO2, 3, 10, 14, 18) and up to 44% (RNO13). Detailed information on all polymorphic and nonpolymorphic markers is listed in Supplementary Table 1.
In our genome scan we identified seven regions (one on RNO2, 3, 5, 10, X and two on RNO13) where two or three consecutive SSLP markers were polymorphic between the DA substrains. We added additional markers in close proximity to those polymorphic regions. For RNO2, RNO5, RNO10, and RNO13 all additional markers were nonpolymorphic, ruling out the possibility of a large contaminating fragment. However, on RNO3 additional markers were found to be polymorphic, suggesting the presence of a large contaminating fragment in this region. It is well known that SSLP regions exhibit a higher mutation rate than the average genome and, thus, the surprisingly high genetic variability could be the result of mutations in the SSLP regions analyzed. However, it is far more likely that it is the result of contamination of one of the DA substrains with another rat strain. To support this theory, we took advantage of the recently performed whole-genome typing of 20,238 SNPs in 167 inbred rat strains, including DA/OlaHsd (Saar et al. 2008). Although the DA/ZtmRhd substrain was not among those strains, a relatively closely related substrain, DA/HanKini, was included. By comparing this strain with DA/OlaHsd we found seven distinct clusters of polymorphic SNPs on five rat chromosomes (Table 1). While six of those regions with sizes between 1 and 4 Mb were considerably small, we found one cluster of SNPs on RNO3 that was more that 10 Mb. We conclude that DA/OlaHsd and DA/ZtmRhd rats differed at a high number of SSLP and at a large contaminating DNA fragment on RNO3.
Fine-mapping of the contaminating fragment on RNO3
List of all arthritis experiments of DA substrains
Incidence of arthritis
Day of onset (mean ± SD)a
Maximal arthritis score (mean ± SD)a
50 μl pristane
12 ± 2
39 ± 11
14 ± 2*
25 ± 11*
50 μl pristane
12 ± 2
46 ± 9
12 ± 1
40 ± 8*
150 μl pristane
13 ± 2
27 ± 14
75 μg CII/100 μl
15 ± 2
36 ± 18
18 ± 3*
21 ± 15*
75 μg CII/100 μl
14 ± 1
53 ± 10
16 ± 2*
33 ± 17*
DA/OlaHsd rat suffered from more severe pristane-induced arthritis
DA/OlaHsd had an earlier onset and increased severity of collagen II-induced arthritis
Several reports of genetically contaminated rat strains and observed phenotypic differences between rats of different colonies have been described earlier. It has been shown that BUF rats from different commercial vendors have polymorphic rat CD45 molecules (Jones et al. 1994) and that Dahl/Rapp rats are polymorphic at the Nos2 gene (Hojna et al. 2005). Furthermore, a polymorphism in the Ncf1 gene in inbred LEW rats from different sources was reported and it was shown that the substrains react differently to the induction of PIA (Olofsson et al. 2004). All these examples clearly show that when it comes to studies in which genetic purity is crucial to the experimental design and interpretation, one should act with caution when comparing animal studies from different institutes.
In arthritis research one of the most commonly used rat strains is the DA rat. The DA rat has a leading role in the search for arthritis-susceptible genes. Worldwide four different institutes have reported linkage studies with collagen II-induced arthritis that were performed using the arthritis-susceptible DA strain in combination with various resistant rat strains such as ACI, BN, E3, and F344 (Dracheva et al. 1999; Griffiths et al. 2000; Gulko et al. 1998; Meng et al. 2004; Olofsson et al. 2003b; Remmers et al. 1996). These studies provide a unique tool for the study of arthritis-regulating loci and for in silico fine-mapping by comparing the QTLs in those linkage studies.
However, caution must be taken so that only different resistant strains are used and not different sources of the susceptible DA rat strain. Here we report the finding of genetic polymorphisms of DA substrains from different origins. The largest contaminating fragment on RNO3 stretches from 35.6 to 52.3 Mb, colocalizing with the previously reported QTL Cia11. Interestingly, the Cia11 QTL was found in two different studies. In the first study by Griffiths et al. (2000), a F2 hybrid of (DA × BN) was used, and it was found that the DA allele from DA/Bkl rats at Cia11 recessively promotes CIA severity. In a different study by Olofsson et al. (2003b), another substrain of DA (DA/ZtmRhd) was used for an F2 backcross with the E3 strain, and in this study the DA allele had a recessive-promoting effect on arthritis severity as well. The Cia11 QTL was found to have a recessive-promoting allele in both DA substrains, but because of their genetic differences on RNO3 between 35.6 and 52.3 Mb we can exclude this region for being responsible for the Cia11 effect. This is confirmed through the comparison of the DA.DA/ZtmRhd-Cia11 congenic rat, which is as susceptible to CIA and PIA as their littermates harboring the DA/OlaHsd allele.
Because we have only limited information on polymorphic markers on RNO3, the origin of the putative contamination remains unclear. SNP data revealed identical haplotypes between ACI and DA/OlaHsd and no match of the DA/HanKini haplotype to any of the included strains, suggesting the contamination originated from ACI. However, previously performed SSLP typing of the recombinant inbred strain DXEC (unpublished data) showed a number of alleles not matching either of the parental strains DA/Ztm and E3/Ztm. Some of these alleles were found to be identical in DA/OlaHsd rats. Additional SNP typing will be required to conclusively determine the origin of the contamination.
In conclusion, we found considerable genetic differences and also major differences in arthritis incidence, severity, and onset of PIA and CIA between the DA/OlaHsd and DA/ZtmRhd substrains. Thus, there must be at least one yet unidentified CIA and PIA locus between those substrains. In our whole-genome scan we found a second region with polymorphic markers colocalizing with a previously described arthritis-regulating region. The polymorphic SSLP markers D10Rat26 and D10Rat93 on RNO10 are considerably close to the confirmed Cia5 and Oia3 QTLs (Brenner et al. 2005; Holm et al. 2001) and to the Pia10 QTL (Olofsson et al. 2003a). Although our additional marker screen or the SNP data did not reveal a large contaminating fragment, there is still the possibility of a minor fragment that differs between the DA/OlaHsd and DA/ZtmRhd substrains, inducing the differences in arthritis severity in those rats. Another possibility is that there is a novel arthritis QTL in one of the other contaminated regions or that the difference in arthritis susceptibility is due to a spontaneous mutation in one of the substrains.
In summary, we demonstrated that DA substrains show distinct genetic differences and exhibited a significantly diverse susceptibility to experimentally induced arthritis. Thus, this study shows how important it is to consider genetic variability in rat inbred strains and that one cannot uncritically compare studies performed with animals of different origin.
We thank the technicians at Medical Inflammation Research (Lund, Sweden), Carlos Palestro and Isabelle Bohlin, for taking excellent care of the animals. We also thank Dr. Lina Olsson and Dr. Robert Bockermann for critically reading the manuscript and for valuable comments. This work was supported by grants from the Swedish Research Council, the Swedish Association against Rheumatism, and the Swedish Foundation for Strategic Research, and European Union Grants MUGEN (LSHG-CT-2005-005203) and EURATools (European Commission contract No. LSHG-CT-2005-019015).